Intermolecular interactions involving proteins include receptor-ligand interactions, receptor-antigen interaction and antibody-antigen interactions. In each case, specific regions of the respective molecules involved in such interactions are involved. Often, regions of proteins involved in intermolecular interactions are loops.
In the case of proteins that are members of the immunoglobulin superfamily, such as antibodies and receptors, loops referred to complementarity determining regions (CDRs) are provided. The differences in sequence of CDRs are generated by alternative splicing of the genes encoding the protein at the region encoding the CDR. This alternative splicing generates a variety of CDRs on different antibodies and thereby allows for the diversity of targets for antibodies. Similarly, different T cell receptors bind to different antigens based upon the diversity of CDR sequences.
Williams, et al., (1988) Annual Review of Immunol., 6:381-405, which is incorporated herein by reference, discloses that the numerous cell surface receptors that belong to the immunoglobulin gene superfamily share certain unique structural features. Antibodies, which are prototypes of the family, are composed of polypeptide chains whose amino acid sequences indicate the presence of homology regions of about 110 amino acids each. These regions fold into compact three-dimensional domains characterized by a .beta.-barrel structure with an internal intradomain disulfide bond spanning approximately 65 amino acids. All members of the immunoglobulin gene superfamily are composed of one or several domains that share varying degrees of amino acid sequence homology. Williams, (1987) Immuno. Today, 8:298-303, which is incorporated herein by reference, discloses that the homology domains are often the product of a single exon.
Kappler, et al., (1987) Cell, 49:263-271, which is incorporated herein by reference, discloses that while the immunoglobulins bind to native antigens, the T cell receptors interact with antigenic fragments that have become associated with either class I or class II proteins which are themselves polymorphic members of the immunoglobulin gene superfamily. Bjorkman, et al., (1987) Nature, 329:506-511, which is incorporated herein by reference, discloses a peptide binding cleft deduced from the atomic structure of certain class I MHC proteins. The sides of the cleft are formed by two .alpha. helices and the bottom surface of the cleft is a .beta. sheet. T cell receptor structures have not been determined but are likely to be similar to immunoglobulins in terms of their tertiary structure. The third hypervariable region of the .alpha. and .beta. polypeptides of the T cell receptor may directly interact with antigen. (See Kappler, et al., supra; Bjorkman, et al., supra; and Fink, et al., (1986) Nature, 321:219, which are incorporated herein by reference.)
Other members of the immunoglobulin gene superfamily include the CD4, CD2 and CD8 molecules. Ryn, et al., (1990) Nature, 348:419-426, and Wang, et al., (1990) Nature, 348:411-418, each of which is incorporated herein by reference, disclose the three-dimensional structure of the protein engineered N-terminal domains of CD4 and CD8. The N-terminal half of the CD4 molecule is folded into two domains stabilized by disulfide bonds, reduction of which impairs the binding of the HIV protein gp120 to CD4. The CD4 domains are analogous to the complementarity determining regions (CDRs) of antibodies. CD4 has CDR2- and CDR3-like regions of anti-parallel .beta. sheets connected by .beta. turns. The CD4 molecule can interact with class II molecules; while the CD8 gene product, which is closely related to light chain gene segments, interacts with class I molecules. Leahy, et al., (1992) Cell, 68:1145-1162, which is incorporated herein by reference, discloses that the solution of the CD8 molecule revealed a very similar structure to that predicted by modeling of the sequence with the REI immunoglobulin structure. It is apparent that the portion of the CD8 molecule that has been analyzed to modest resolution (2.6 .ANG.) is quite comparable with the CD4 structure as well. It has been possible to model which of the CDR loops of the CD8 molecule might bind to the class I structure. Modeling results suggest the centrally placed CDR2 loop attaches to the class I molecule. Likewise, using a different approach Fleury, et al., (1991) Cell, 66:1037-1049, which is incorporated herein by reference, has suggested that the CD4 CDR1 and CDR3 loops might be relevant to class II binding.
Fleury, et al., supra; Clayton, et al., (1988) Nature, 335:363-366; and Konig, et al., (1992) Nature, 356:796-798, each of which is incorporated herein by reference, disclose that part of the problem in studying large protein surface interactions by Alanine or site-directed mutagenesis is that introduction of hydrophobic or hydrophilic residues into the protein main chain at certain positions produces major conformational changes that cannot be anticipated without structural corroboration.
One study (Salter, et al., (1990) Nature, 345:41-46 which is incorporated herein by reference) has implicated a discrete loop of the .alpha.-3 domain of the class I molecule as part of the binding site for CD8, while Konig, et al. (1992) Nature, supra and Cammarota, et al., (1992) Nature, 356:799-800, which are incorporated herein by reference, have suggested an analogous structural motif on the .beta.-2 domain of the class II molecule as a target for binding the CD4 ectodomain. Therefore, it is likely that both CD4 and CD8 use CDR-like loops to bind to other loop-like projections on the respective targets. Although T cell receptors can function in the absence of these CD4 or CD8 structures, they appear to play a critical role in some level of activation and ligation. In addition, they may also be important in some aspects of T cell development.
Amit, et al., (1986) Science, 233:747-753, which is incorporated herein by reference, discloses that molecular and crystallographic analysis of immunoglobulins has revealed that the critical ligand binding surfaces are CDR projections. In addition, it is apparent that there are canonical conformations of the complementarity determining regions of the V kappa light chain CDRs and two of the heavy chain CDRs. The third CDR of the heavy chain, as a consequence of the complex genetic mechanism which influences its structure, has medium or long loops which have diverse patterns of interactions. In general, the canonical CDRs, aside from the CDR3 of the heavy chain, have reverse turn conformations which can sometimes have the regular features of turns. (See Saragovi, et al. (1992) Biotechnology.) In addition, Williams, et al., (1988) Annual Review of Immunol., 6:381-405 which is incorporated herein by reference, and Williams, Immuno. Today, supra, discloses that it is apparent that the C1 and C2 types of domains, while similarly fashioned from two .beta. sheets linked together by a disulfide bond, serve as models of other .beta. loop types and further disclose that the C1 domains are involved in antigen interactions, while C2 domains subserve Fc receptor and adhesive structures such as LFA-3, MAG, CD2 and NCAM and ICAM.
The conformational properties of peptide loops or reverse turns are considered important mediators in the biological activity of polypeptides. Turns provide for suitable orientations of binding groups essential for bioactivity by stabilizing a folded conformation in a small molecule and may be involved in both binding and recognition sites. See for example: Saragovi, et al., (1986) Science, 233:747-753; Chen, et al., (1992) Proc. Nat'l Acad. Sci. U.S.A., 89:5872-5876; and Sibanda, et al., (1989) J. Mol. Biol., 206:759-777, each of which is incorporated herein by reference. The studies of small naturally occurring peptides, such as somatostatin and encephalins, emphasized the role of turns in the optimal placement of side-chains for receptor binding and the influence of backbone conformations.
The field of synthetic peptides is one of intense activity, particularly the design of synthetic peptides useful to mimic biologically active proteins. A great deal of effort has been expended to identify the portion of a protein which is directly involved in intermolecular activity and to model small peptides based upon the amino acid sequence of that portion. Synthetic peptides are designed which are modelled upon the active regions of proteins. Such synthetic peptides may consist of identical sequences as that of the sequence of the protein which is involved in intermolecular interactions or they may comprise additional flanking amino acid sequences and/or include additions, deletions and/or substitutions within the sequence.
Linear synthetic peptides that are designed based upon the active portions of biologically active molecules, particularly loops, and more particularly CDRs, have achieved limited success. The biological activity of linear peptides which are designed based include regions that identical or substantially similar to active portions of biologically active molecules are often less than that of the biologically active protein. The reason for the diminished activity is that the linear peptides are not conformationally stable and shift from active to inactive conformations. Linear peptides are characteristically highly flexible molecules whose structure is strongly influenced by their environment, and their random conformation in solution may preclude their practical application to mediate binding and biological effects. Linear peptides often assume an aggregated state rather than an intramolecular folded state. It has been suggested that high conformational flexibility of small linear peptides and the volume to surface ratio are not favorable for proper folding (Marshall, et al. (1978) Ann. Rep. Med. Chem., 13:227-238, which is incorporated herein by reference) and that this tendency precludes the use of short peptides as defined biological or therapeutic agents (Saragovi, et al., (1992) Biotechnology).
To address this reduction in activity, it is often desirable to provide conformationally restricted peptides. Conformationally constrained peptides which contain the biologically active loop have been designed and synthesized to provide peptides with loop regions which are conformationally restricted. Peptides are cyclicized or otherwise constrained by peptide or non-peptide bonds in order to maintain the active region in a stable and active conformation.
Williams, et al., (1991) J. Biol. Chem., 266:5182-5190, and Williams, et al., (1991) J. Biol. Chem., 296:9241-9250, each of which is incorporated herein by reference, describe the isolation and synthesis of conformationally constrained peptides derived from the complementarity determining regions of the light chain of antibodies. These constrained loops were analyzed to define the atomic basis of interaction of the individual residues with respect to binding. Four critical side chains at the tip of the loop were found to project into space and hydrogen bond with the target on the cell surface or to antibodies to which they bound. In addition, it was shown that it is possible to use the CDRs from immunoglobulins to develop constrained macrocyclic loops that have biological and antigen binding activities. Anti-receptor antibodies were utilized as a source of complementarity determining regions loop structures since the antibodies trigger a discrete biochemical response in cells upon ligating the receptor. In one set of studies, constrained peptides derived from the second CDR of the light chain of an anti-receptor antibody were shown to lead to an inhibition of lymphocyte DNA synthesis much like other immunologically active immunosuppressants.
Several examples of CDRs from anti-receptor antibodies acting as the major attachment sites of the antibodies were also disclosed. In these cases of anti-receptor antibodies, the residues of the antibody framework may be less important than in other foreign antigen binding immunoglobulins. This may be a consequence of the selection strategy of anti-receptor antibodies as opposed to antibodies to foreign antigens. The criteria for selecting anti-receptor antibodies is that they mediate a biological role independent of the binding properties. Consequently, selection is less biased by affinity considerations since even moderate affinity anti-receptor antibodies are adequate for most studies. In contrast, antibodies specific for soluble foreign antigens are usually selected for high affinity interactions.
Certain cyclic peptides are disclosed which demonstrate enhanced binding when compared to the corresponding linear peptides. These observations are consistent with the fact that critical ligand binding surfaces of immunoglobulins and related proteins are in a reverse turn conformation. Therefore, if a linear peptide is predicted to be more active in a turn configuration since it was derived from a known loop in the original protein structure, it can be subjected to cyclization. Cyclization can be readily achieved by the incorporation of cysteine residues during peptide synthesis, followed by oxidation. An intramolecular covalent disulfide bond is thus created which restricts the configuration of the peptide. An important consideration is the size or diameter of the loops obtained by cyclization. In order to develop insights into the diameter of the CDR loop studies were undertaken to constrain the loop and the orientation of the side chains. In one 16-mer, cysteine residues were introduced at random places to achieve a system of constrained loops. The cysteines placed at the 9th and 16th positions were far less effective at cyclization than cysteines placed in the 10 and 16th position. This indicates that small errors in spatial positioning can create compounds with reduced binding and biological activity. In addition, the constraint of the appropriately looped structures resulted in 40-fold higher levels of affinity than linear ones.
There is a need for improved synthetic peptides. There is a continue need for a means of increasing the biological activity of synthetic peptides designed based upon active regions of biologically active proteins. There is a need for conformationally restricted peptides which demonstrate improved biological activity. There is a need for conformationally restricted peptides which have enhanced affinity to the molecules that they interact with.